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. Author manuscript; available in PMC: 2016 Jan 1.
Published in final edited form as: Oncogene. 2014 Sep 29;34(29):3848–3859. doi: 10.1038/onc.2014.315

Figure 5.

Figure 5

LKB1 deficiency promotes DNA oxidation and mutagenesis. (a) LKB1 depletion increases 8-oxo-dG. U2OS/shR-Ctrl and U2OS/shR-LKB1 cells growing on fibronectin-coated coverslips were treated with 500 μM H2O2 in serum-free and phenol-red-free medium for 2 h at 37°C, fixed in absolute methanol (− 20°C, 20 minutes) and permeabilized with 0.1% Triton X-100 at room temperature for 15 min. After blocking, the cells were stained with FITC-conjugated avidin for 1 h at 37°C. Fluorescence is captured with an Olympus IX51 fluorescence microscope. (b) Levels of 8-oxo-dG in LKB1-depleted cells treated with p38 inhibitor and ROS scavenger. U2OS/shR-Ctrl and U2OS/shR-LKB1 cells cultured in 96-well plate were pre-treated with 10 μM SB203580 or 5 mM NAC for 2 h before exposure to 500 μM H2O2 for additional 2 h. After the treatment, the cells were fixed in 4 % paraformadehyde and permeabilized with 0.1 % triton X-100 at room temperature for 15 min. The cells were washed with PBS for 3 times and stained with FITC-conjugated avidin for 1 h at 37°C. The supernatants were discarded and 100 ul PBS per well was added for analysis by fluorescence multi-well plate reader with excitation and emission wavelengths of 495 nm and 525 nm. Intensity of fluorescence is expressed as the mean ± SEM. *P < 0.01; N.S., not significant (n = 3). (c) Levels of 8-oxo-dG in SKOV3/shR-Ctrl and SKOV3/shR-LKB1 cells insulted by H2O2. Measurement of 8-oxo-dG was performed as in b. Intensity of fluorescence is expressed as the mean ± SEM. *P < 0.01 (n = 3). (d) Mutations within HPRT locus in U2OS/shR-Ctrl and U2OS/shR-LKB1 cells. One million cells were treated with 1.35 μM 6-TG. Three weeks later, the growth colonies were fixed with 4 % paraformaldehyde and stained with crystal violet. (e) p38 reduces LKB1-deficiency-induced mutation. HPRT assays were performed as in d in U2OS/shR-Ctrl and U2OS/shR-LKB1 cells stably transfected with pcDNA3 (vector), p38-WT, and p38-CA. Mutation frequencies within HPRT locus were calculated. The number of colonies is expressed as the mean ± SEM. *P < 0.01 (n = 3).